Phosphorylation of mitochondrial proteins is a rapid and efficient way to regulate oxidative phosphorylation (OXPHOS) and maintain energy homeostasis. Mitochondrial PKA modulates enzymes of the electron transfer chain through protein phosphorylation, which is regulated by a signaling pathway, involving mitochondrial soluble adenylyl cyclase (sAC), generating cAMP that activates PKA. This signaling cascade serves as a metabolic sensor that modulates energy conversion in mitochondria. Cytochrome oxidase (COX) is a pacemaker of ETC fluxes, whose activity is modulated by phosphorylation of its subunits and by ATP allosteric inhibition. We showed that COX is a target of the signaling pathway and identified subunit IV of COX (COXIV) as a target for phosphorylation. We also demonstrated that phosphorylation of S58 in COXIV- 1 is responsible for modulation of COX activity. We propose that this regulatory pathway participates to metabolic adaptive responses to OXPHOS defects and could be a target for pharmacological intervention. The goal of this project is to understand the physiological implications of intramitochondrial sAC-cAMP- PKA signaling and S58 COXIV-1 phosphorylation, in vivo. We will understand how this system behaves in healthy tissues and in tissues affected by mitochondrial defects. To this end, we will generate and study mice, in which the molecular players of the regulatory pathway are genetically modified and assess the outcomes on clinical phenotype and mitochondrial biochemistry. In aim 1, we will generate transgenic mice expressing inducible sAC selectively targeted to the mitochondrial matrix (mito-sAC), which is expected produce high basal metabolism. In aim2, we will generate COXIV-1 S58A knockin mice, lacking the phosphorylated site, and thus incapable of up-regulating ETC fluxes and enhancing ATP production, resulting in exercise intolerance, decreased glucose utilization, impaired thermogenesis, and increased fat storage. In aim 3, we will investigate sAC-cAMP-PKA modulation in a mouse model of OXPHOS defect caused by conditional and inducible genetic disruption of COX. These mice recapitulate biochemical and clinical characteristics of mitochondrial diseases. We will assess how sAC-cAMP-PKA modulation and protein phosphorylation in mitochondria from affected tissues and correlate it with disease progression. PUBLIC HEALTH RELEVANCE: We identified a cAMP-driven signaling pathway for protein phosphorylation within mitochondria that works as a metabolic sensor. We will investigate this metabolic regulation by the mitochondrial cAMP-dependent phosphorylation pathway in health and disease, by studying how the components of the pathway affect the metabolic responses in genetically modified mouse models. A better understanding of the function of this mitochondrial pathway of metabolic regulation will help to unravel the pathogenic mechanisms of metabolic defects in human diseases and their treatment.